Myofibroblastic activation of hepatic stellate cells (HSCs) is presumed to play a central role in liver fibrogenesis, and the mechanism underlying this process is a potential therapeutic target for liver fibrosis. However, different mesenchymal cell populations give rise to myofibroblasts and participate in liver fibrogenesis, and their relationships to HSCs are yet to be clarified. We believe understanding of liver mesenchymal cell lineages is a prerequisite for gaining critical insights into the mechanisms underlying the generation of myofibroblasts in liver fibrosis. Using genetics-based cell lineage analysis, we have demonstrated that the liver mesothelium migrates inward from the liver surface and gives rise to HSCs and perivascular fibroblasts during liver development. Based on this novel finding, we have begun our investigation on the possible contribution of the liver mesothelial cells (MCs) to liver fibrosis in adults. Our preliminary data support that MCs migrate inward and differentiate into myofibroblasts during liver fibrosis induced by CCl4 or alcohol treatment in mice. Isolated MCs from fibrotic livers, express increased levels of epithelial-mesenchymal transition (EMT) driver (Snail1 and 2) and marker (SMA, type I collagen) genes. Further MCs from the normal liver undergo EMT into myofibroblasts by TGF-2. Based on these findings, we propose a ground-breaking hypothesis that the MC serves as a novel source of myofibroblasts in alcoholic liver fibrosis and as a potential therapeutic target for the disease. To test our hypothesis, 1) we will determine the lineage relationship and relative contribution of liver MCs and HSCs to alcohol-induced liver fibrosis using MC- or HSC-specific Cre mice crossed with Rosa26flox mice. These genetic models will label and quantitatively trace MCs and HSCs in the genesis of myofibroblasts in different anatomical locations (submesothelial, perisinusoidal, periportal, and perivenular) during alcohol-associated liver fibrogenesis in an animal model given alcohol and a diet high in cholesterol and saturated fat. 2) Using newly developed transgenic reporter mouse lines, we will isolate MCs and HSCs from alcoholic liver fibrosis and control liver, to perform comparative gene profiling and to test the role of EMT by a loss or gain of function manipulation for EMT genes (Snail1 or Tgfbr2) in culture. 3) We will selectively target deletion of these EMT genes in MCs in the model of alcoholic liver fibrosis using MC-specific Gpm6a promoter-Cre mice with a floxed target gene to test its therapeutic effect. 4) Finally, we will validate the expression of key markers for MC EMT, which are shown to be important in mouse models, in patients with alcoholic liver fibrosis. PUBLIC HEALTH RELEVANCE: Chronic alcohol consumption causes alcoholic liver disease which is the second most common gastrointestinal cause of death in the United States. The proposed study will determine how the liver surface tissue actively participates in alcohol-induced liver fibrosis as a new mechanism. The knowledge obtained from this study will help lead to the development of novel and efficacious modalities aimed at promoting liver regeneration and suppressing liver fibrosis.